Project 3 - Mapping Long-range Allosteric Pathways in CRISPR-Cas9

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Project Summary Gene regulatory mechanisms are critical for proper cellular and protein function, and modern molecular biology has linked numerous pathologies to dysregulation of these processes. Although modification of the genome to correct pathogenic mutations is a promising therapeutic approach, these efforts cannot be successful without knowledge of the underlying biochemistry of protein machinery such as CRISPR- Cas9 (Cas9). Cas9 can be a customizable tool to edit and correct disease-linked (genomic) mutations, however, to fully realize these applications, novel strategies to overcome its off-target effects and poor temporal control must be investigated. Cas9 utilizes a guide RNA molecule to recruit, stabilize, and facilitate cleavage of double-stranded DNA after recognition of a well-known protospacer adjacent motif (PAM) sequence. Prior X-ray crystal structures indicate that conformational changes within the Cas9 nucleases, HNH and RuvC, are required for effective catalytic function. However, these structures offer little mechanistic information, as the target DNA and catalytic nucleases are never observed in an activated state. The conformational shift of HNH, in particular, is correlated to motions of neighboring subdomains, all of which are activated from >20 Å away by the PAM-binding domain, suggesting an allosteric mechanism. Understanding this allosteric coupling would have exciting potential for precision medicine by establishing novel paradigms to control and enhance the spatial and temporal function of Cas9. We recently identified a pathway of millisecond timescale motions spanning the HNH nuclease and reaching multiple Cas9 domains that computational results suggest is a portion of a larger allosteric network that controls Cas9 function. To investigate the reach of this allosteric network and the role of molecular motions in its mechanism, my laboratory will undertake a synergistic solution NMR and computational study to map the long-range allosteric pathway of Cas9. We will now (1) characterize allosteric mutants of HNH that are known to alter Cas9 specificty, (2) establish the biophysical roles of the neighboring REC2 and REC3 domains in propagating allosteric signals to/from HNH, and (3) characterize the conformational ensemble governing the full-length Cas9 protein. This multidisciplinary approach of NMR spin relaxation experiments, molecular dynamics simulations, and network theory, will probe multi-timescale protein motions in Cas9, revealing specific amino acids responsible for transmitting structural or dynamic information. These studies will use both full-length Cas9 and novel engineered constructs to interrogate specific domains within the 160 kDa enzyme. The structural and dynamic findings of this work will be correlated to function with new in vivo assays to provide a detailed understanding of the Cas9 allosteric mechanism.